JoTT Re v i e w 3(8): 1975–1980

Emerging trends in molecular systematics and molecular phylogeny of (Insecta: Ephemeroptera)

K.G. Sivaramakrishnan 1, K.A. Subramanian 2, M. Arunachalam 3, C. Selva Kumar 4 & S. Sundar 5

1 Flat No.3, Rams Apartments, No.7 Natesan Street, T. Nagar, Chennai, Tamil Nadu 600017, India 2 Zoological Survey of India, Western Regional Station, P.C.N.T. Post, Rawet Road, Akurdi, Pune, Maharastra 411044, India 3,4,5 Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, Tamil Nadu 627412, India Email: 1 [email protected], 2 [email protected] (corresponding author), 3 [email protected], 4 [email protected], 5 [email protected]

Date of publication (online): 26 August 2011 Abstract: Current trends are reviewed in the molecular systematics and phylogeny of the Date of publication (print): 26 August 2011 Ephemeroptera (mayflies), an ancient monophyletic lineage of pterygote . Theories ISSN 0974-7907 (online) | 0974-7893 (print) of origins are analyzed, followed by a discussion of higher classification schemes Editor: V.V. Ramamurthy in light of recent developments in molecular systematics. Ephemeroptera evolution is a classic example of ancient rapid radiation, presenting challenges for phylogenetic analysis. Manuscript details: The utility of combined studies of morphological and molecular data is substantiated with Ms # o2661 examples and the role of molecular systematics in unraveling the of cryptic Received 29 December 2010 Final received 21 July 2011 species complexes is highlighted. The importance of DNA barcoding in mayfly taxonomy Finally accepted 29 July 2011 is discussed in the light of recent progress, and future contributions of genetics to the study of taxonomy, ecology and evolution in mayflies are discussed. Citation: Sivaramakrishnan, K.G., K.A. Subramanian, M. Arunachalam, C.S. Kumar & Cryptic species, DNA barcoding, Ephemeroptera, molecular phylogeny, S. Sundar (2011). Emerging trends in molecular Keywords: systematics and molecular phylogeny of molecular systematics. mayflies (Insecta: Ephemeroptera). Journal of Threatened Taxa 3(8): 1975–1980. Copyright: © K.G. Sivaramakrishnan, K.A. INTRODUCTION Subramanian, M. Arunachalam, C. Selva Kumar & S. Sundar 2011. Creative Commons Attribution 3.0 Unported License. JoTT allows unrestricted use of this article in any medium The order Ephemeroptera presently encompasses over 3000 species for non-profit purposes, reproduction and and 400 genera, constituting at least 42 described families (Barber-James distribution by providing adequate credit to the authors and the source of publication. et al. 2008). The Ephemeroptera (mayflies) are an archaic lineage of

Author Detail: see end of this article. insects, dating back to the late Carboniferous or early Permian periods,

Author Contribution: KGS and KAS some 290 mya (Brittain & Sartori 2003). They occupy freshwater and conceived and prepared the review. MA actively participated in the discussion of preparation of brackish water habitats across the world, with the exception of Antarctica. the manuscript and provided critical inputs. CSK The nymphs are immature stages inhabiting lentic and lotic waters. and SS helped in compiling the literature. The imagos or adults are terrestrial; they lack mouth parts and do not Acknowledgements: K.G.Sivaramakrishnan is grateful to Dr. M. Arunachalam for having feed, relying on nutritional build up during immature stages. They invited him to Sri Paramakalyani Centre for have an ephemeral lifespan of a day or two and their only function is Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, reproduction. The presence of a subimago with functional wings at the Tamilnadu, India. He is indebted to authorities of Manonmaniam Sundaranar University for penultimate moult is unique to pterygote insects. The winged stages of having offered facilities to carry out a UGC Ephemeroptera, as with Odonata (dragonflies and damselflies) and the Major Project, during which period he could interact with co-authors to organize this review. extinct Palaeodictyoptera, cannot fold their wings horizontally over the K.A.Subramanian is grateful to the Director, Zoological Survey of India for providing facilities abdomen as neopterans can. to prepare the manuscript. This article briefly reviews current trends in the molecular systematics and phylogeny of the Ephemeroptera and discusses how combined analysis of morphological and molecular data can be used to fine tune phylogenetic conclusions.

Mayfly origins

OPEN ACCESS | FREE DOWNLOAD The phylogenetic position of Ephemeroptera within the winged insects

Journal of Threatened Taxa | www.threatenedtaxa.org | August 2011 | 3(8): 1975–1980 1975 Mayfly systematics and phylogeny K.G. Sivaramakrishnan et al.

(Pterygota) is hotly debated by systematists, and Costatergalia, which is equal to McCafferty’s (1991) significant disagreement still exists in morphological + Setisura + Oniscigastridae. Topological and molecular studies. The first complete mitochondrial comparison of Kluge’s system and McCafferty’ system genome of a heptageniid mayfly, Parafronurus youi of mayfly classification is presented in Fig. 1, after was sequenced using a long PCR-based approach Ogden et al. (2009). In contrast to previous hypotheses by Zhang et al. (2008). In their analysis, the basal based on morphological observations, the relationships Ephemeroptera hypothesis (Ephemeroptera versus inferred from the molecular data (Ogden & Whiting (Odonata + Neoptera)) was supported. This result also 2005) were congruent in some cases, but incongruent received strong support by the nucleotide and amino in others. In their analysis, the groups, Furcatergalia, acid datasets from mitochondrial protein-coding , Carapacea, and Caenoidea genes with BI and ML analyses. Zhang et al. (2008) and 15 families were supported as monophyletic. tentatively concluded that mitochondrial genomes can On the other hand, Setisura, Pisciforma, Baetoidea, answer the difficult question of the basic relationships Siphlonuroidea, Ephemeroidea, Heptagenoidea and among the winged insects. Ephemeroptera evolution five families (having more than one taxon represented) is a classic example of “ancient rapid radiation of were not supported as monophyletic. insects” presenting challenges for phylogenetic However, evidence supports the notion that analysis because such radiations take place over short combined data (morphology + molecular data) periods of time and allow few distinctive phylogenetic analysis provides a more robust estimate of markers to accumulate among lineages. phylogenetic relationships. The study of Ogden et The Ephemeroptera, Odonata and Neoptera present al. (2009) represents the first formal morphological a challenging phylogenetic tree shape, regardless of and combined (morphological and molecular) their true relationships, because the first pterygotes phylogenetic analyses of the order Ephemeroptera. may have emerged up to 400 mya, but the earliest Taxonomic sampling comprised 112 species in 107 representatives of their extant descendants is much genera, including 42 recognized families (all major younger than the first emergence of the lineage whose lineages of Ephemeroptera). Morphological data relationships are in question (Whitfield & Kjer 2008). consisted of 101 morphological characters. Molecular data were acquired from DNA sequences of 12S, 16S, Molecular systematics and higher 18S, 28S and H3 genes. The Asian genus Siphluriscus classification (Siphluriscidae) was supported as sister to all other mayflies. The lineages Carapacea, Furcatergalia, The original subordinal classification of McCafferty Fossoriae, Pannota, Caenoidea and Ephemerelloidea & Edmunds (1979), based mostly on thoracic were supported as monophyletic. However, some morphology and wing pad position, comprised the recognized families (for example, Ameletopsidae holophyletic suborder Pannota and the paraphyletic and ) and major lineages (such as suborder Schistonota indicating the retention of certain Setisura, Pisciforma and Ephemeroidea among others) plesiomorphic (ancestral) traits. It was realized that were not supported as monophyletic, mainly due to monophyly derived from synapomorphy (shared convergences within nymphal characters (Ogden et al. derived characters) should be the driving force behind 2009). any taxonomic classification (Hennig 1966, 1979; Farris 1979). Later, McCafferty (1991) proposed 3 different Efficacy of combined morphological suborders (Pisciforma, Setisura and Rectracheata) and and molecular phylogeny and traced phylogenetic relationships within and among the systematics - examples from the suborders. Concurrent to McCafferty’s work, Kluge Ephemeroptera (1988, 1998) independently proposed two suborders for Ephemeroptera. His suborder Furcatergalia is It is quite obvious that most previous reconstructions equivalent to McCafferty’s Rectracheata, except the of phylogeny and classification were strongly hampered exclusion of Oniscigastridae form Furcatergalia. by superficial external morphological similarities, The other suborder proposal (Kluge 1988) was which do not always reflect the true phylogeny of the

1976 Journal of Threatened Taxa | www.threatenedtaxa.org | August 2011 | 3(8): 1975–1980 Mayfly systematics and phylogeny K.G. Sivaramakrishnan et al.

Figure 1. Topological comparison of Kluge’s system and McCafferty’s system of mayfly classification (after Ogden et al. 2009).

Order. Homoplasies therefore seem a dominant trait 69 leptophlebiids sampled from six continents and in mayfly morphology and behaviour, especially in representing 30 genera plus 11 taxa of uncertain nymphs (Ogden et al. 2009) and combined analysis taxonomic rank from Madagascar and Papua New may solve many riddles. Apart from the outstanding Guinea. Although they did not recover monophyly recent contribution of Ogden et al. (2009) on these of the , monophyly of two of the lines towards evolving a new paradigm in mayfly three leptophlebiid subfamilies, Habrophlebiinae and phylogeny, some families notably, Leptophlebiidae Leptophlebiinae, was recovered with moderate to (O’Donnell & Jockusch 2008), (Gattolliat et strong support in most analyses. The Atalophlebiinae al. 2008) and (Ogden et al. 2009) have was rendered paraphyletic as a result of the inclusion received considerable attention regarding intrafamilial of numbers of Ephemerellidae or the Leptophlebiinae relationships, in which molecular phylogenetic tools clade. For the species-rich Atalophlebiinae, four were extensively employed. groups of taxa were recovered with moderate to strong Using two nuclear genes (the D2 + D3 region of branch support: (i) an endemic Malagasy clade, (ii) 28S ribosomal DNA and histone H3) and maximum a Paleoaustral group, a pan-continental cluster with parsimony (MP), maximum likehood (ML) and members drawn from across the Southern Hemisphere, Bayesian inference (BI), O’Donnell and Jockusch (iii) the Choroterpes group uniting fauna from North (2008) inferred the evolutionary relationships of America, southeast Asia and Madagascar and (iv) a

Journal of Threatened Taxa | www.threatenedtaxa.org | August 2011 | 3(8): 1975–1980 1977 Mayfly systematics and phylogeny K.G. Sivaramakrishnan et al. group uniting three new world genera, Thraulodes, do not allow reliable distinction. This is particularly Farrodes and Traverella. true among larvae, the life-stage used most widely in Gattolliat et al. (2008) reconstructed the biomonitoring studies. Williams et al. (2006) assessed first comprehensive molecular phylogeny of the the molecular diversity of this complex in one of the Afrotropical Baetidae. They sequenced a total of ca. largest such studies of cryptic species in the order 2300 bp from nuclear (18S) and mitochondrial (12S Ephemeroptera to date. Phylogenies were constructed and 16S) gene regions from 65 species belonging to using data from the mitochondrial cytochrome oxidase 26 genera. They used three different approaches of submit I (COI) gene. Two monophyletic groups were phylogeny reconstruction viz., direct optimization, recovered consisting of one major haplogroup and a maximum parsimony and maximum likelihood. The second clade of six smaller but distinct haplogroups. molecular reconstruction indicated the Afrotropical Haplogroup divergence ranged from 0.2–3 % (within) Baetidae require a global revision at a generic as well to 8–19 % (among) with the latter values surpassing as suprageneric level. maxima typically reported for other insects, and The investigation of Ogden et al. (2009) represented provided strong evidence for cryptic species in the the combined molecular and morphological analysis for B. rhodani complex. However, the taxonomic status the mayfly family Ephemerellidae (Ephemeroptera), of these seven haplogroups remains to be defined with a focus on the relationships of genera and clearly. species groups of the subfamily Ephemerellinae. The The potential implications of cryptic species in the phylogeny was constructed based on DNA sequence B. rhodani complex on current and future ecological data from three nuclear (18S rDNA, 28S rDNA, studies are particularly far-reaching given the large histone H3) and two mitochondrial (12S rDNA, 16S number of studies carried out on what now appears rDNA) genes, and 23 morphological characters. to be several possible distinct taxa. These results , the largest genus of Ephemerellidae, have wider relevance since cryptic species have been and Serratella were not supported as monophyletic detected in other aquatic insects (Jackson & Resh lineages. Strongly supported as monophyletic 1998), and the presence and diversity of several taxa include a grouping of the Timpanoginae genera are widely used as biological indicators (Mason 1996). Timpanoga, Dannella, Dentatella and Eurylophella, The presence of cryptic species also has ramifications and groupings of the Ephemerellinae genera Torleya, for the assessment of biodiversity in general, and the Hyrtanella and Crinitella and the genera Kangella, ability to account for them in future studies emphasizes Uracanthella and Teloganopsis. Further study and the need to correlate genetic differences from multi- analysis of Ephemerellidae morphology is needed, locus data, with identifiable morphological characters and classification should be revised, if it is to reflect and/or other factors including physiology. true phylogenetic relationships (Ogden et al. 2009). Dna barcoding and mayfly taxonomy Molecular systematics and cryptic species complex The tool of DNA barcoding shows great potential for use by those studying the systematics of many Genetic studies have highlighted cryptic diversity Ephemeroptera species groups. One example of in many well-known taxa including aquatic insects, the utility of barcoding is the verification of stage with the general implication that there are more associations, especially those not made by careful species than are currently recognized. Baetis rhodani rearing. Recent revisionary study, on the family Pictet are among the most widespread, abundant Ephemerellidae Klapalek provides an illustration. The and ecologically important of all European mayflies species concept of Ephemerella altana Allen, a western (Ephemeroptera), and used widely as biological Nearctic taxon, had been based on a larva belonging to indicators of stream quality. Traditional taxonomy the genus Ephemerella Walsh and an adult of Serretella and systematics have never fully resolved differences Edmunds. Had barcoding technology been available among suspected cryptic species in the B. rhodani at the time of E. altana’s discovery and description, it complex because morphological characters alone potentially could have shown that this association was

1978 Journal of Threatened Taxa | www.threatenedtaxa.org | August 2011 | 3(8): 1975–1980 Mayfly systematics and phylogeny K.G. Sivaramakrishnan et al. erroneous. Furthermore, barcoding could have helped primary applications of genetic techniques. Application to resolve the species identities of the larva and adult. of the Generalized Mixed Yule-coalescent (GMYC) Based on traditional specimen comparisons, the larva model to species circumscription using single-locus is thought to be that of the transcontinental species, DNA appears rewarding (Pons et al. 2006; Fontaneto et Ephemerella excrucians Walsh, and the adult to be that al. 2007). This approach has been applied successfully of the western species, Serretella micheneri (Traver). to 64 species of mayflies in Madagascar (Monaghan et Ephemerella excrucians exhibits an amazing amount al. 2009). of morphological variability throughout its wide - Investigators of the demographic history of geographic range, which begs the question of whether closely related populations or species can use several the current species concept might contain various nuclear DNA sequences to test specific hypotheses cryptic lineages that are unrecognizable by traditional, of how past geological events influence observed morphological means. Barcoding technology could patterns (e.g. Knowles et al. 2007). These techniques be used to study various populations, including those allow one to test a priori hypotheses rather than post from type localities, and could provide a guideline for hoc conclusions from patterns (Monaghan & Sartori decisions about species identities and boundaries (Zhou 2009). et al. 2008). Zhou et al. (2009) have made a pioneering - Wilcock et al. (2005) demonstrated very well how attempt to generate a DNA barcode reference library the combination of ecological and genetic research, for three orders- Ephemeroptera, Plecoptera and applied to several parts of the life cycle, can greatly Trichoptera at one site in the Canadian subarctic. This advance our understanding of how populations study has demonstrated that DNA barcoding holds great function in nature. promise as a tool for rapid biodiversity assessment in - Routine sampling of population- and species- level unknown faunas. A very close correspondence was genetic diversity, combined with coalescent-based observed between morphospecies as determined by methods of species delineation has great potential taxonomic experts and barcode clusters designated to become a standard procedure for the study of using a standard sequence threshold. Several cases of poorly known taxonomic groups like Ephemeroptera proposed splitting may reflect cryptic species. (Gattolliat & Monaghan 2010). DNA barcodes of stream mayflies will improve descriptions of community structure and water quality for both ecological and bioassessment REFERENCES purposes (Sweeney et al. 2011). Rapid assessment of biodiversity will aid the selection of sites of special Barber-James, H.M., J.L. Gattolliat, M. Sartori & conservation value and will help to focus the efforts M.D. Hubbard (2008). Global diversity of mayflies (Ephemeroptera: Insecta) in freshwater. Hydrobiologia 595: of taxonomists in revising and characterizing the 339–350. diversity of life (Zhou et al. 2009). Brittain, J.E. & M. Sartori (2003). Ephemeoptera of Insects. (Edited by Resh, V.H. & R.T. Carde). Academic Press, San Diego, 373–380pp. CONCLUSIONS Caterino, M.S., S. Cho & F.A.H. Sperling (2000). The current state of insect molecular systematics: a thriving tower of Babel. Annual Review of Entomology 45: 1–54. Future perspectives on systematics and phylogeny Farris, J.S. (1979). On the naturalness of phylogenetic of Ephemeroptera using recent molecular tools are classification.Systematic Zoology 28: 200–214. highlighted below: Fontaneto, D., E.A. Herniou, C. Boshetti, M. Caprioli, G. - Four sequencing markers which are well-surveyed Melone, C. Ricci & T.G. Barraclough (2007). Independently and informative across a range of divergences viz., the evolving species in asexual bdelloid rotifers. PLoS Biology mitochondrial COI and 16S genes, and the nuclear 5: 914–921. Gattolliat, J.L. & M.T. Monagan (2010). DNA-based 18S and EF-Iα genes are suggested as standards for association of adults and larvae in Baetidae (Ephemeroptera) comparison for insect molecular systematic studies with the description of a new Adnoptilum in Madagascar. (Caterino et al. 2000). Journal of the North American Benthological Society 29(3): - Species delineation continues to be one of the 1042–1057. (http://www.bioone.org/doi/abs/10.1899/09-

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Frontiers in Annals of the Entomological Society of America 84: 343– Zoology 6: 30 (doi: 10.1186/1742-9994-6-30). 360. Monaghan, M.T. & M. Sartori (2009). Genetic contributions to the study of taxonomy, ecology, and evolution of mayflies Author Details: Dr. K.G. Si v a r a m a k r i s h n a n has been working on aquatic (Ephemeroptera): review and future perspectives. Aquatic insects especially on the systematics and biogeography of mayflies Insects 31: 19–39. (Ephemeroptera) of India over last 35 years. He has many publications on the subject in international and national journals. Currently he is based Monaghan, M.T., R. Wild, M. Elliot, T. Fujisawa, M. Balke, in Chennai. D. J.G. Inward, D.C. Lees, R. Ranaivosolo, P. Eggleton, Dr. K.A. Su b r a m a n i a n is a scientist at Zoological Survey of India, Kolkata and has been working on aquatic insects since 1998. T.G. Barraclough & A.P. Vogler (2009). Accelerated species Dr. M. Ar u n a c h a l a m Professor at Sri Paramakalayni Centre for discovery on Madagascar using a coalescent-based model of Environmental Sciences, Manonmaniam Sundaranar University, species delineation. Systematic Biology 58: 298–311. Alwarkuruchi. He is a freshwater biologist and Ichthyologist and has been working on ecology and systematics of freshwater organisms, especially O’Donnell, B. & E.L. Jockusch (2008). Phylogenetic the freshwater fishes. relationships of leptophlebiid mayflies as inferred by histone C. Se l v a Ku m a r Doctoral student at Sri Paramakalayni Centre for Environmental Sciences, Manonmaniam Sundaranar University, H3 and 28S ribosomal DNA, Systematic Entomology 33: Alwarkuruchi. Currently working on Ephemeroptera of Kalakkad- 651–667. Mundanthurai Tiger Reserve. S. Su n d a r Doctoral student at Sri Paramakalayni Centre for Environmental Ogden, T.H. & M.F. Whiting (2005). Phylogeny of Sciences, Manonmaniam Sundaranar University, Alwarkuruchi. Currently Ephemeroptera (mayflies) based on molecular evidence. working on Naucoridae (Hemiptera) of southern Western Ghats.

1980 Journal of Threatened Taxa | www.threatenedtaxa.org | August 2011 | 3(8): 1975–1980